Global Methylation Profiling by Selective Release of Methylated Sites from Immobilized Tryptic Peptides

Methylation
of lysine and arginine (K and R) has emerged
as a prevalent
post-translational modification with critical roles in numerous biological
processes. The current identification approaches suffer from suboptimal
enrichment efficiency, particularly for lysine methylation, hindering
comprehensive KR methylome profiling. Herein, we presented an antibody-free
strategy termed Selective Release of Methylated Sites from Immobilized
Tryptic Peptides (SRMs-ITP), which achieves high enrichment efficiency
while enabling the simultaneous analysis of all five methylation states
of KR. This strategy exploits the unique ability of LysargiNase to
cleave methylated KR residues, which are absent in trypsin-based digestion.
Totally, our approach identified 5516 methylation sites across 2866
proteins from HeLa cell lysate, including 2405 arginine methylation
sites and 3111 lysine methylation sites. SRMs-ITP achieved an enrichment
efficiency exceeding 48.2%, significantly outperforming current antibody-based
and antibody-free strategies. Notably, 56.4% of the detected methylation
sites were on lysine residues, surpassing the existing antibody-free
approaches. These findings establish SRMs-ITP as a robust, unbiased,
and highly efficient methodology for KR methylome analysis. The approach
offers a powerful tool for deciphering the intricate regulatory mechanisms
of protein methylation and its cross-talk with other post-translational
modifications under various physiological and pathological conditions.